Project Summary/Abstract Combination Antiretroviral Therapy (cART) has been tremendously effective in HIV-infected patients by suppressing HIV replication and maintaining the virus at undetectable levels. However, although cART is life- prolonging, it never eradicates HIV infection or cures this disease. Interruption of treatment quickly results in virus replication and recurrence of the disease. As a result, HIV-infected patients have to receive lifelong cART treatment which presents economic and therapeutic burdens. To this end, a ?shock and kill? strategy has been proposed to reactivate (?shock?) latent HIV in the presence of cART and immune-based therapy to purge (?kill?) these reservoirs. However several serious issues surrounding this strategy limit its clinical application. Therefore there is an urgent need to develop novel effective therapeutic strategy. Recently a ?soothe and snooze? strategy has been proposed to permanently silence the latent provirus by stronger and more durable virus-suppressing agents. Didehydro-cortistatin A (dCA), a specific inhibitor for HIV-1 Tat, a key HIV viral trans-activating regulatory protein, has been shown to prevent HIV-1 reactivation from latency. In this project, we seek to develop novel and more effective drugs to silence the latent HIV provirus, based on our knowledge on epigenetic regulation of HIV gene transcription. It has been established that elongation is the rate limiting step of HIV gene transcription. We hypothesize that inhibition of elongation will block HIV provirus gene expression, resulting in deep viral suppression. The super elongation complex (SEC) is critical for HIV transcription elongation. In our preliminary study, we have shown that the core component of the SEC, the AFF proteins, contain novel epigenetic ?reader and ?writer? (HAT) activities, making them ideal therapeutic targets. We will apply the cutting edge DNA-encoded chemistry technology (DEC-Tec) to perform large scale screening to identify small molecule inhibitors of AFF1 and AFF4. Such inhibitors would silence the HIV provirus, and combination of AFF inhibitors and Tat inhibitor dCA might further suppress HIV activation to achieve a functional cure for HIV-1 infection. Our proposal will be carried in two phases. In R61 phase of the first three years, the goal is to identify AFF1 and AFF4 inhibitors. Aim 1, to identify small-molecule compounds that bind to AFF1/4 by DNA-encoded chemistry technology. Aim 2, to test if these compounds inhibit HIV transcription and AFF function. In R33 phase of the last two years, the goal is to perform functional characterization of AFF1/4 inhibitors. Aim 3, validation of inhibitory activity of AFF1/4 inhibitors on HIV transcription in primary cell model and combination therapy with Tat inhibitor dCA. Aim 4, pharmacokinetics (PK) and toxicity studies of AFF1/4 inhibitors.